Lafb LBYBI13 Microscopy PDF

Week 01 MICROSCOPY Leo Agustin F. Barcelo, MSc., RMicro Department of Biology De La Salle University LBYBI13 | Microscopy | 01  OBJECTIVES 1. Understand the basic principles of microscopy and how th...

Week 01 MICROSCOPY Leo Agustin F. Barcelo, MSc., RMicro Department of Biology De La Salle University LBYBI13 | Microscopy | 01  OBJECTIVES 1. Understand the basic principles of microscopy and how they work 2. Identify the different kinds of microscope and their applications and limitations 3. Understand key terms in used in microscopy such as magnification, resolution Transmission Electron Microscope Image source: https://www.dlsu.edu.ph/research/central-instrumentation-facility/tem/ LBYBI13 | Microscopy | 02  WHAT IS A MICROSCOPE? Microscope A lab instrument used to view and examine objects that are too small to be seen by the naked eye. Hans Janssen A compound microscope and its parts LBYBI13 | Microscopy | 03  HOW DOES IT WORK? Light coming from a light source (mirror or bulb) will travel through the specimen. The light hitting the specimen will be focused (concentrated) by a lens or set of lenses called objectives The focused light will now travel the tube/body through the eyepiece which further increases the magnification. How a light microscope magnifies an object LBYBI13 | Microscopy | 04  THE VIRTUAL IMAGE The virtual image is the final image that an observer sees when viewing a microscope It can be affected by many factors, which can also be manipulated to get better quality images. Some of these factors are: The type of light (electromagnetic spectrum) used The properties or size of the object being observed Lens type and placement Contrast Electromagnetic Spectrum LBYBI13 | Microscopy | 05  KEY PRINCIPLES OF MICROSCOPY Magnification - The degree on how a device can enlarge an object/specimen. In microscopes, this is provided by the lens. - It is also written in the lens as “X” e.g. 40X, 10x etc. Eyepiece lenses (magnifying power indicated) (Objective magnification) x (Eyepiece magnification) = Total magnification LBYBI13 | Microscopy | 06  KEY PRINCIPLES OF MICROSCOPY Resolution - Ability of a microscope to differentiate two adjacent objects as discrete entities - wavelength of light and numerical aperture (NA) wavelength Resolving power = 2 x (NA) Numerical aperture - dictates the light gathering ability of the objective and the condenser LBYBI13 | Microscopy | 07  KEY PRINCIPLES OF MICROSCOPY Refractive index - Measure of the bending of light as it passes from one medium to another - Lights bends as it passes through the glass side which can cause light dispersion - In higher magnification, oil can be used to lessen dispersion as it has same index as glass. Light dispersion with oil (top) vs. with oil (bottom) Light enters the Better resolution Light refracted objective LBYBI13 | Microscopy | 08  KEY PRINCIPLES OF MICROSCOPY Illumination - Light source e.g., mirror or light bulb Condenser - collects the light from the light source and form into a cone of light that is focused on the specimen Diaphragm - Used to adjust the size of the opening (aperture) in which the light passes through Iris diaphragm (left) and condenser (right) before reaching the condenser LBYBI13 | Microscopy | 09  KEY PRINCIPLES OF MICROSCOPY Parfocality of Microscopes - During observation, most microscope objectives can be switched with minimal refocusing - Parfocal lenses maintain focus even when focal length is changed. Contrast - Interaction of the light and the specimen - Adding contrast between the specimen and the background can aid in the viewing the details of the specimen. LBYBI13 | Microscopy | 10  TYPES OF MICROSCOPE Two-dimensional Imaging Bright Field Microscope - specimens are visualized because of differences in contrast (density) between the specimen and surrounding. - Uses two set of lenses (objective and ocular) - Maximum magnification ~ 2,000X LBYBI13 | Microscopy | 11  TYPES OF MICROSCOPE Two-dimensional Imaging Phase Contrast Microscope - Uses a phase ring which can induce phase shifts due to brightness variation - creates contrast without the use of dyes - Live samples can be observed - Dark cells on light background LBYBI13 | Microscopy | 12  TYPES OF MICROSCOPE RBCs under a brightfield microscope (left) and by a phase contrast microscope (right) LBYBI13 | Microscopy | 13  TYPES OF MICROSCOPE Two-dimensional Imaging Dark Field Microscope - Light from light source is blocked of thus light reaches the specimen from the side - Best for observing motility RBCs under a dark field microscope LBYBI13 | Microscopy | 14  TYPES OF MICROSCOPE Two-dimensional Imaging Fluorescence Microscope - Visualize specimens that fluoresce (emit light when it absorbs other wavelengths of light) - Fluorescence can be natural or induced (through staining e.g. using DAPI) - Used in microbial ecology for Cells under a fluorescence microscope stained enumeration of bacterial population using DAPI (left) and eosin (right) LBYBI13 | Microscopy | 15  TYPES OF MICROSCOPE Three-dimensional Imaging Differential Interference Contrast (DIC) Microscopy - Uses polarizer w/c creates two distinct beams of polarized light - The polarized light can make some cell structure (e.g. vacuole, endospores, etc.) appear 3D Image generated from a DIC microscope LBYBI13 | Microscopy | 16  TYPES OF MICROSCOPE Three-dimensional Imaging Confocal Scanning Laser Microscope (CLSM) - Computerized microscope + laser source - The computer focuses the laser on single layers of the specimen - Resolution is 0.1 μm Image of a mini brain organoid generated from a CLSM Image Source: https://directorsblog.nih.gov/tag/confocal-laser-scanning-microscope/ LBYBI13 | Microscopy | 17  TYPES OF MICROSCOPE Electron Microscope Transmission Electron Microscope (TEM) - Electromagnets serves as lenses - High magnification and high resolution (0.2 nm) - Requires a vacuum and specimens should be sliced thinly (20-60 nm) - Requires staining e.g., uranyl acetate and lead citrate Orthopoxvirus under a TEM. Bar = 200 nm Image Source: https://www.rki.de/EN/Content/infections/Diagnostics/diagnostics_node.html LBYBI13 | Microscopy | 18  TYPES OF MICROSCOPE Electron Microscope Scanning Electron Microscope (SEM) - Beams of electron hits heavy metal coated specimen - Gold is a common heavy metal coat used - The electrons that will scatter will hit the detectors rendering an image - Magnification 15x to 100,000 x Spirochete observed using an SEM Image Source: https://api.semanticscholar.org/CorpusID:18208022 LBYBI13 | Microscopy | 19  - END - GUIDE QUESTIONS FOR THIS TOPIC IS UPLOADED IN CANVAS. If this topic piqued your interest, you can check out this video for a more in depth discussion of electron microscopy. https://www.youtube.com/watch?v=LVDokjhQrl4&t=2637s LBYBI13 | Microscopy | 20